Container with at least one electrode

ABSTRACT

The invention relates to a container  20, 30  for receiving an aqueous solution, which is formed at least partially by an outer limit  21  forming an inner chamber  22, 32  for receiving the solution, and which comprises at least one area which acts as an electrode  25, 26, 33, 34  when an electric voltage is applied and a subsequent discharge occurs, wherein at least one electrode  25, 26, 33, 34  is made of a conductive synthetic material at least based on a plastic material which is doped with at least one conductive substance. A container  20, 30  of the above-mentioned kind is created this way, which is simple and economical to produce and also, for example, enables an efficient transfection of living cells by means of electroporation or an effective electrofusion.

The invention concerns a container for receiving an aqueous solution,and in particular cells, derivatives of cells, subcellular particlesand/or vesicles, which is formed at least partially by an outer limitforming an inner chamber for receiving said solution, and whichcomprises at least one area which acts as an electrode when an electricvoltage is applied and a subsequent discharge occurs.

BACKGROUND OF THE INVENTION

Transferring biologically active molecules, such as, for example, DNAs,RNAs or proteins, into living cells is an important tool for analysis ofbiological functions of these molecules. Electroporation is a preferredmethod for transferring foreign molecules into the cells, which incontrast to chemical methods causes less undesirable changes of thebiological structure and function of the target cell. Duringelectroporation the foreign molecules are introduced into the cells froman aqueous solution, preferably a buffer solution adapted to the cellsor a cell culture medium, by a short-time current flow, i.e. the pulseof a discharging capacitor, whereby the cell membrane is made permeablefor the foreign molecules by effect of the short electric pulses.Solution and cell suspension, respectively, are usually provided in aso-called cuvette, i.e. a small container which is open at the top andwhich comprises two oppositely and parallel arranged electrodes disposedin the sidewalls near the bottom and serving for the application ofelectric voltage. Through the temporarily emerging “pores” in the cellmembrane the biologically active molecules initially reach the cytoplasmwhere they eventually already exert their function to be analysed. Atcertain conditions the molecules subsequently also enter the nucleus ofthe cell. Particularly with the introduction of DNA into animal cells,the so-called transfection, specific problems often arise duringelectroporation because of the fragility of the cells since theefficiency of transfection is affected by the survival rate of the cellsas an important parameter.

Due to the temporarily applied intensive electric field, i.e. a shortpulse with high current density, cells, derivatives of cells,subcellular particles and/or vesicles can also be fused. During thisso-called electrofusion at first, for instance, the membranes of thecells are brought in close contact by an inhomogenous alternatingelectric field. The subsequent application of an electric field pulseleads to an interaction of parts of the membranes, which finally leadsto cell fusion. Comparable devices such as the ones used forelectroporation can be used for electrofusion as well.

State of the Art

Containers as mentioned above are known and primarily used forelectroporation or electrofusion in the form of cuvettes having insertedelectrodes made of metal. Containers used for this purpose are mostlysmall vessels which are closed at the bottom and open at the top andwhose inner space is build by two pairs of parallel and oppositelyarranged sidewalls. The inner space serves for receiving of the cellsuspension, i.e. usually an aqueous buffer solution or a cell culturemedium, in which the cells to be treated are suspended. Such cuvettesmostly comprise a pair of electrodes for application of an electricvoltage disposed near the bottom of a pair of oppositely arrangedsidewalls. During an electric discharge an electric current flowsthrough the cell suspension between both electrodes, which enables anintroduction of nucleic acids or other molecules into the cells or,depending on the selected conditions, leads to fusion of cells. Theelectrodes are mostly made of metal, wherein aluminium is frequentlyused. But it is an disadvantage of these known, commercially availablecuvettes that metal ions are emitted into the buffer solution during theelectric discharge, which can cause an undesirable stimulation of thecells at lower concentrations or, at higher concentrations, act toxic onthe cells. For instance, using cuvettes made of aluminium a negativeeffect due to the release of Al³⁺ ions could be demonstrated(Loomis-Husselbee et al., Biochem J 1991, 277 (Pt 3), 883-885).Furthermore, using cuvettes having electrodes made of metal generationof undesirable precipitates may occur, which are also generated due tothe release of metal ions from the electrodes. The precipitates may bemetal hydroxides or complexes of metal ions with biologicalmacromolecules of the buffer solution (Stapulionis, BioelectrochemBioenerg 1999, 48(1), 249-254). Finally, it is another disadvantage ofcuvettes made of aluminium that the resistance of the cuvettes decreasesduring discharge, presumably because a layer of oxidized aluminiumhaving a higher resistance is released from the electrode by the currentflow. Additionally, cuvettes having electrodes made of metal aredifficult to produce and very expensive.

U.S. Pat. No. 6,001,617 discloses a device for cultivation of cells,which can be used for electroporation as well as for electrofusion ofcells. The device consists of a round container having an opticallytransparent bottom on which a layer of cells can adhere and grow. Thebottom of the container may consist of an optically transparent,non-conductive material being coated with an electrically conductivematerial, or can be completely made of an optically transparent andelectroconductive material. The electroconductive bottom is contactedvia a band-like electrode made of metal and being circumferentiallydisposed in the wall area. To act as counter electrode a likewisecircular band is provided. It is one drawback of this known device thatit is primarily provided and suitable for electroporation of adheringcells. Using this device for transfection of suspended cells is limitedand merely possible if low efficiency is accepted. Due to the ring-likecircular contacting of the bottom electrode and the counter electrodebeing likewise cercumferentially disposed in the wall area a homogenouselectric field cannot be generated so that equal transfection of allcells cannot be achieved. This effect is increased by the fact that theintrinsically conductive plastics used have a higher resistance as theyare provided as thin coating. Thus, the cells adhering to the bottom inthe centre of the surface can only be transfected with very lowefficiency. Furthermore, because of the sophisticated construction andthe fact that the intrinsically conductive plastics used are notmouldable, the production of the known container is very expensive.

Description of the Invention

It is thus the object of the invention to create a container asmentioned above in order to overcome the existing deficiencies, whichcan be produced easily and cost-effectively, and which allows anefficient treatment of cells, derivatives of cells, subcellularparticles and/or vesicles using electric current.

According to the invention the object is solved in that at least oneelectrode is made of a conductive synthetic material at least based on aplastic material which is doped with at least one conductive substance.To avoid the release of metal ions a conductive synthetic material beingmade of doped plastic is used. Thus, toxic effects, for instance onliving cells, such as during the release of Al³⁺ ions can be avoided.Medical compatibility of the generating products can also be enhancedthis way so that, for example, the possible use of transfected primarycells for ex vivo gene therapy is supported. As a result of doping theplastics with conductive substances, a current flow between bothelectrodes could be achieved during discharge, which is equal to thecurrent flow using commonly used cuvettes having metal electrodes.Surprisingly, it has been determined that using the used doped plasticmaterial with electroporation better results in respect of thetransfection efficiencies can be achieved if compared to the use of, forinstance, cuvettes having electrodes made of aluminium. By avoiding thetoxic effects caused by the release of metal ions the ratio oftransfection efficiency to cell mortality is significantly increased. Itis a further advantage of the container according to the invention thatno precipitate is created in the solution during electric discharge,which could adhere to the cells and impair further analysis and use oftransfacted cells, respectively. Obviously, it is yet a furtheradvantage of the container according to the invention that the buffersolution has less effect on the doped plastic electrodes used. It wasactually determined that in the cause of discharge very high resistanceis generated at the surface of the electrode, which, at a given startcurrent, causes a drastical decrease of current flow, if bufferscontaining phosphate but no chloride are used with cuvettes made ofaluminium. Surprisingly, this can be avoided by using the container andelectrodes according to the invention, respectively. The containersaccording to the invention are thus merely affected by the conductivityof the solution but not negatively affected by the buffers used. Thecontainers according to the invention can be injection-moulded in onemould using two-component injection moulding because doped syntheticmaterial, in contrast to intrinsically conductive plastic, isinjection-mouldable. Thus, the container according to the invention canbe produced in an easy and cost effective manner. The doped plasticmaterial may have a density, for example, between 1.3 and 1.5 g/cm³,preferably a density of 1.37 or 1.54 g/cm³, and a melting temperature of230 to 310° C. Preferably, the doped plastic material has a specificforward resistance of 2 Ohm×cm or less, and a specific input resistanceof 10⁴ Ohm or less.

In respect of the conductivity of the synthetic material it turned outto be particularly advantageous if the dope consists of carbon fibers,graphite, soot, carbon nanotubes and/or an intrinsically conductivesynthetic material. The overall concentration of the dope in the plasticmaterial should be in the range between 10 and 80 % w/w. Theconductivity of the synthetic material is not sufficient below apercentage of 10%, while the possibility to injection-mould thesynthetic material is extremely reduced above a percentage of 80%. Usingthe container according to the invention with electroporation orelectrofusion of cells, derivatives of cells, subcellular particlesand/or vesicles it has been shown that it is particularly advantageousin respect of conductivity that the overall concentration of the dope is20-60% w/w, more preferred 40-60% w/w, particularly preferred 50-60%w/w, in particular 55-60% w/w.

Several applications, in particular transfection of DNA into the nucleusof primary cells, need particularly high electric currents and fieldstrengths, respectively, in the solution to achieve sufficiently highefficiencies. In these cases it is advantageous if the overallconcentration of the dope in the plastic material is between 40 and 80 %w/w. This ensures a high conductivity of the electrodes so that flow ofsufficiently high currents through the solution and the inner space ofthe container, respectively, is possible. In an advantageous embodimentof the invention the concentration of the dope may be in the range of50-80% w/w, preferably 60-80% w/w, more preferred 70-80% w/w, inparticular 74-76% w/w, depending on the application and the type of thecells to be treated. A sufficient injection-mouldability of the materialis ensured in this respect even if the dope has high concentrations upto 80% w/w. In this respect, the use of particular mixtures of dopes,e.g. carbon fibers and graphite, may have a positive influence onmouldability.

In respect of the injection-mouldability of the synthetic material itturned out to be particularly advantageous if the plastic material ispolycarbonate, polyetheretherketone, polypropylene, polyamide,polyphenylensulfide or a mixture of these polymers, or at least based onone or several of these polymers, and/or wherein said plastic materialis an intrinsically conductive synthetic material. In this respect, theuse of polyamide 66 or polyamide 6 is particularly advantageous.

If the synthetic material itself is doped with an intrinsicallyconductive synthetic material, an advantageous embodiment of theinvention is provided, wherein the intrinsically conductive syntheticmaterial is polyaniline, polyacetylene, poly-para-phenylene,poly-para-phenylensulfide, polypyrroles, polythiophene, polypropylene orthe like, or at least based on one or several of these polymers.

In another advantageous embodiment of the invention it is provided thatthe outer limit is made of synthetic material, preferably transparentplastic material, because it is thus possible to produce the entirecontainer using injection moulding methods. It may be advantageous inthis respect if the outer limit is made of the same plastic material asthe plastic material on which the at least one electrode is based. Inthis embodiment there may be benefits with the processing of thesynthetic material using two-component injection moulding. Thereby, onthe one hand the production is simplified and on the other hand theproduction costs are reduced. However, for special applications, theouter limit may consist of other material as well.

In a particularly advantageous embodiment of the invention it isprovided that the at least one electrode is integrated into said outerlimit so that the container can be injection-moulded in one mould.

In a preferred embodiment of the invention it is provided that thecontainer comprises at least two electrodes being made of the samematerial. Most applications use containers and cuvettes, respectively,which have two oppositely arranged, parallel electrodes consisting ofthe same material. These two electrodes are contacted in a suitablemanner and thereby connected to a voltage source adapted to therespective requirements. However, in special cases it may also bebeneficial if at least two electrodes are made of different materials.In these cases, the anode or the kathode may consist of the dopedsynthetic material while the respective counter electrode may consist ofanother material, e.g. stainless steel or an intrinsically conductivesynthetic material.

The containers comprising electrodes made of a conductive syntheticmaterial according to any one of the claims 12 to 18 proved to beparticularly advantageous for transfection of living cells. Thesecontainers have a high conductivity combined with electrode materialwhich is easliy processable.

In an alternative embodiment of the invention it is provided that theouter limit comprises at least one opening for supplying the solutionand at least one opening for draining off the solution. The containeraccording to the invention can thereby also be used as flow-throughcontainer with which the solution flows through the inner chambercontinously or discontinously.

The containers according to the invention are advantageously suitable inthe form of container arrangements comprising at least two, preferably6, 12, 24, 48, 96 or more, containers being joined to build one unit,i.e. so-called “multi-wells”.

It is a particular advantage of the invention that the containers orcontainer arrangements according to the invention can be produced usinga method, wherein the container or the container arrangement is producedby two-component injection moulding, wherein at first the outer limit isinjection-moulded leaving one recessed window and the conductivesynthetic material being made of doped plastic is subsequentlyinjection-moulded into this at least one window, or wherein at first theat least one electrode is injection-moulded of the doped plasticmaterial and the outer limit is subsequently injection-moulded aroundthe at least one electrode. Contrary to the production of containers orcuvettes with electrodes consisting of metal, with which the electrodeshave to be manually or automatically placed into the moulded frame ormould before or after moulding of the container, the invention providesa very easy and cost-effective method of production. The containers orcontainer arrangements according to the invention can beinjection-moulded in one mould in a two-step method so that the costs oftheir production are significantly lower as the usual costs for theproduction of electroporation or elektrofusion cuvettes.

Advantageously, the containers or container arrangements according tothe invention are particularly suitable for use in methods for treatmentof cells, derivatives of cells, subcellular particles and/or vesicles bymeans of electric current, in particular for electroporation orelectrofusion, wherein the cells, derivatives of cells, subcellularparticles and/or vesicles are transferred into an inner chamber of atleast one container or at least one container of a containerarrangement, wherein said container comprises at least one electrodebeing made of a doped synthetic material, and at least one furtherelectrode, and wherein a voltage is then applied to said electrodes anda current flow is generated in the inner chamber of said container. Inthis method, the electric current may reach a current density up to 120A/cm², preferably 80 A/cm², and the cells, derivatives of cells,subcellular particles and/or vesicles may be provided in suspension, oradhered or in otherwise immobilized condition.

Accordingly, beside other possible applications, the containers orcontainer arrangements according to the invention are suitable, forinstance, for electroporation, i.e. methods for introducing biologicallyactive molecules into living cells using electric current, whereinbiologically active molecules, in particular nucleic acids, are solvedin said solution, and transfer of said biologically active moleculesinto living cells is achieved by means of a voltage pulse having a fieldstrength of 2 to 10 kV*cm⁻¹ and a duration of 10 to 200 μs.Subsequently, a current flow following said voltage pulse withoutinterruption, having a current density of 2 to 14 A*cm⁻², preferably 5A*cm⁻², and a duration of 1 to 100 ms, preferably 50 ms, can beprovided. In this method, the cells can be used, for example, insuspension or as an adhering cell layer.

The containers or container arrangements according to the invention arealso suitable, for instance, for electrofusion, e.g. methods for fusionof cells, derivatives of cells, subcellular particles and/or vesiclesusing electric current, wherein the cells, derivatives of cells,subcellular particles and/or vesicles, for example, are suspended at asuitable density in an aqueous solution, said suspension is subsequentlytransferred into a container or container arrangement according to theinvention, and finally an electric voltage is applied to the electrodesgenerating a current flow through the soultion. Alternatively, forexample, also adhering cells, derivatives of cells, subcellularparticles and/or vesicles can be fused as well as, for example, adheringcells with suspended cells, derivatives of cells, subcellular particlesor vesicles.

The invention is described below in detail with reference to thedrawings.

In the figures

FIG. 1 is a perspective representation of an experimental assembly fordemonstration of the present invention,

FIG. 2 shows the current flow of electric discharges using electrodesaccording to the invention, which are made of polycarbonate including20% carbon fibers and 15% graphite (PC+CF+Gr having an electrodethickness of 1.65 mm), compared to the usage of electrodes made ofaluminium, respectively with two different buffer solutions (Solution A:100 mM sodiumphosphate, pH 7.1, and 25 mM potassiumchloride; Solution B:140 mM sodiumphosphate, pH 7.1; Ordinate: current, 1 A per square;Abscissa: time, 10 ms per square),

FIG. 3 is a perspective representation of an experimental assemblyaccording to FIG. 1 modified by insertion of copper wires,

FIG. 4 shows the current flow of electric discharges using electrodesmade of polycarbonate including 20% carbon fibers (Electrode thickness 2mm), and using an experimental assembly according to FIG. 1 (a) and FIG.3 (b), ordinate: current, 1 A per square and abscissa: time, 10 ms persquare,

FIG. 5 shows diagrams of a flow-cytometric analysis of transfected CHOcells (Chinese hamster ovary cells) using different electrodes made ofsynthetic material according to the invention compared to electrodesmade of aluminium, a) number of transfected cells per 25,000 cells, b)percentage of dead cells stained with propidiumjodide,PC/CF/Gr=polycarbonate+20% carbon fibers+15% graphite with an electrodethickness of 1.65 mm, PEEKIDF=polyetheretherketone+40% carbon fiberswith an electrode thickness of 1 mm,

FIG. 6 shows diagrams of a flow-cytometric analysis of transfected HL-60cells (human lymphoma cells) using different electrodes made ofsynthetic material according to the invention compared to electrodesmade of aluminium, a) number of transfected cells per 15,000 cells, b)percentage of dead cells stained with propidiumjodide,PC/CF/Gr=polycarbonate+20% carbon fibers+15% graphite with an electrodethickness of 1.65 mm, PEEK/DF=polyetheretherketone+40% carbon fiberswith an electrode thickness of 1 mm,

FIG. 7 shows diagrams of a flow-cytometric analysis of transfectedJurkat cells (human T-cell line) using electrodes made of syntheticmaterial according to the invention compared to electrodes made ofaluminium, a) number of transfected cells per 25,000 cells, b)percentage of dead cells stained with propidiumjodide, PA/CF=polyamide66+30% carbon fibers with an electrode thickness of 1 mm,

FIG. 8 shows diagrams of a flow-cytometric analysis of transfected HUVECcells (human umbilical cord vein endothelial cells) using electrodesmade of synthetic material according to the invention compared toelectrodes made of aluminium, a) number of transfected cells per 15,000cells, b) percentage of dead cells stained with propidiumjodide,PPS/CF=polyphenylensulfide+40% carbon fibers,

FIG. 9 is a micrograph of CHO cell cultures (CHO=Chinese hamster ovarycells) 2 days after electroporation using a) electrodes made ofaluminium and b) electrodes made of polyphenylensulfide+40% carbonfibers,

FIG. 10 shows diagrams of a flow-cytometric analysis of HUVEC cells(human umbilical cord vein endothelial cells) after incubation indifferently treated solutions, a) number of dead cells stained withpropidiumjodide, b) number of living cells stained withcarboxyfluoresceindiacetate-succinimidylester (CFDA-SE), PA/CF=polyamide66+30% carbon fibers with an electrode thickness of 1 mm,PC/CF/Gr=polycarbonate+20% carbon fibers+15% graphite with an electrodethickness of 1.65 mm,

FIG. 11 shows diagrams of a flow-cytometric analysis of CHO cells(Chinese hamster ovary cells) after incubation of differently treatedsolutions, a) number of dead cells stained with propidiumjodide, b)number of living cells stained withcarboxyfluoresceindiacetate-succinimidylester (CFDA-SE),PP/CF=polypropylene+20% carbon fibers with an electrode thickness of 1mm, PPS/CF=polyphenylensulfide+40% carbon fibers, PA/CF=polyamide 66+30%carbon fibers with an electrode thickness of 1 mm,

FIG. 12 is a perspective representation of one possible embodiment ofthe container according to the invention,

FIG. 13 is a sectional (a) and a perspective (b) representation of afurther embodiment of a container according to the invention in the formof a cuvette,

FIG. 14 shows diagrams of a flow-cytometric analysis of HL-60 cells(human lymphoma cells) after electroporation using electrodes made ofsynthetic material according to the invention (PA6) compared toelectrodes made of aluminium, a) number of dead cells stained withpropidiumjodide, b) number of transfected living cells,untreated=respectively without application of a voltage pulse,

FIG. 15 shows diagrams of a flow-cytometric analysis of CD3 ⁺ T-cellsafter electroporation using electrodes made of synthetic materialaccording to the invention (PA6) compared to electrodes made ofaluminium, a) number of dead cells stained with propidiumjodide, b)number of transfected living cells, respectively with or without vectorpH2-K^(k),

FIG. 16 shows diagrams of a flow-cytometric analysis of HUVEC cells(human endothelial cells) after electroporation using electrodes made ofsynthetic material according to the invention (PA6) compared toelectrodes made of aluminium, a) number of dead cells stained withpropidiumjodide, b) number of transfected living cells,untreated=respectively without application of a voltage pulse, and

FIG. 17 shows diagrams of a flow-cytometric analysis of HL-60 cells(human lymphoma cells) 24 and 96 hours after electroporation usingelectrodes made of synthetic material according to the invention (PA66and PA6) compared to electrodes made of aluminium, a) number of deadcells stained with propidiumjodide, b) number of transfected livingcells, untreated=respectively without application of a voltage pulse.

FIG. 1 shows a perspective representation of an experimental assembly 1for demonstration of the present invention and for testing of thecontainers according to the invention, respectively. The experimentalassembly 1 is equivalent to the construction of the containers accordingto the invention and comprises a spacer plate 2 and two electrodes 3, 4being pressed against both sides of the spacer plate 2. The spacer plate2 is a plate made of Teflon having a thickness of 2 mm, which has anU-like formed recess 5. The electrodes 3, 4 consist of a syntheticmaterial which is doped with at least one conductive substance. Thesynthetic material may be, for example, polycarbonate,polyetheretherketone, polypropylene, polyamide, polyphenylensulfide or amixture of these polymers and/or an intrinsically conductive syntheticmaterial. The three layers which include the spacer plate 2 and bothelectrodes 3, 4 are pressed together at both sides like a sandwich bythe copper plates 6, 7. For this purpose, the copper plates 6, 7 aremoved towards the other by the threaded parts 8, 9 of a vise-like device(not shown). When the above-named layers are pressed together an innerspace 10 is then formed in the range of the recess 5, which has thecapacity to receive the solution and cells, respectively. In thedepicted model the inner space 10 can receive a volume of 100 ml. Theinner space 10 is further formed at the bottom and at both sides by theinner edges 11 of the spacer plate 2, and at the two remaining sides bythe electrodes 3, 5. The copper plates 6, 7 are electrically contactedvia two wires to spring contacts of a customary electroporation device,i.e. a voltage source. The electrodes 3, 4 themselves are in directcontact to the copper plates via their entire outer surface so that thebest electric contact is ensured. Thus, using the experimental assembly1 as shown, a current flows between the electrodes 3, 4 through theinner space 10 filled with the solution and the cell suspension,respectively, if a voltage pulse is applied to the copper plates 6, 7.

FIG. 2 shows the current flow of electric discharges using electrodesaccording to the invention, which are made of polycarbonate with 20%carbon fibers and 15% graphite (PC+CF+Gr), compared to the use ofelectrodes made of aluminium. In this approach, the current flow throughtwo different buffer solutions was measured (Solution A: 100 mMsodiumphosphate, pH 7.1 and 25 mM potassiumchloride; solution B: 140 mMsodiumphosphate, pH 7.1). A first voltage pulse of 1000 V and 40 μsduration was always applied followed without interruption by a secondpulse having a start voltage (U_(start)) of 90 V and a charge of 75 mC.The respective current flow of the second pulse is shown in thisrepresentation. Surprisingly, it turned out that, if the electrodes madeof synthetic material according to the invention are used, a similarcurrent flow of that scale can be achieved under the same conditions ascan be achieved with the customary electrodes made of aluminium(comparisons a and c). The doped synthetic material is thus particularlysuitable for conducting high current densities in a short period oftime. Using a phosphate buffer without chloride (Solution B) in contrastto the use of a phosphate buffer containing chloride (Solution A) itturned out that in the course of discharge a very high resistance isincreasingly generated at the surface of electrodes made of aluminium,which causes a drastic decrease of current flow at the same startcurrent (b). This negative effect does not occur when doped syntheticmaterial is used (d) so that, contrary to usually used cuvettes, thecontainers or electrodes according to the invention are also suitablefor the use of phosphate buffers without chloride. Thus, using thecontainers according to the invention the selection of the buffersolution is not as limited as with the use of conventional containers.

FIG. 3 shows a perspective representation of an experimental assembly 12which is modified in comparison to the experimental assembly 1 accordingto FIG. 1. The experimental assembly 1 according to FIG. 1 shows anarrangement where the electrodes 3, 4 are contacted by the copper plates6, 7 using relatively high pressure on a large surface. Since contactingof the outside of electrodes, for example with a conventionalelectroporation apparatus, is usually not carried out with such a highpressure on such a large surface, in this experimental assembly 12 thesurfaces contacting the electrodes are designed smaller. Thus, thisassembly is more equivalent to a contacting by spring contacts and henceto the actual conditions in reality. For this purpose, round and v-likebended copper wires 13, 14 having a diameter of about 1.5 mm were placedbetween the electrodes 3, 4 and the respective adjacently arrangedcopper plates 6, 7. In the following, FIG. 4 shows a comparison of thecurrent flows if the experimental assemblies according to FIG. 1 and 3are used.

FIG. 4 shows the current flow of electric discharges using electrodesmade of polycarbonate including 20% carbon fibers for the experimentalassembly 1 according to FIG. 1 (a) as well as for the experimentalassembly 12 according to FIG. 3 (b). The current flows of the secondpulse are always shown (1. Pulse: 100 V, 40 ps; 2. Pulse: Ustart=90 V,75 mC). The constant result shows that obviously the electric potentialis steadily distributed on the inside of the electrodes and thecontacting surface is thus not the limiting factor in respect ofconductivity.

FIG. 5 shows diagrams of a flow-cytometric analysis of transfected CHOcells. Transfection experiments are conducted using the experimentalarrangement described in FIG. 1 in order to biologically determine thefunctionality of the containers according to the invention. For thispurpose, CHO cells were suspended in 100 μl of an appropriate buffersolution, for example PBS (phosphate-buffered saline), adding 5 μg ofthe expression plasmid pH-2K^(k) (DNA vector which codes for the heavychain of a mouse MHC class I protein), and then transferred into theinner space of the experimental assembly. Electroporation of the cellsis then carried out by application of two pulses (1. Pulse: 1000 V, 100ps; 2. Pulse Ustart=108 V, 100 mC). The cell suspension was subsequentlyremoved, transferred into an appropriate medium, for example RPMImedium, and harvested after 20 hours incubation at 37° C. and 5% CO₂.Adhering CHO cells were washed with PBS and detached using 0.1%trypsine+2 mM ethylendiamintetraacetate in PBS. The expression ofH-2K^(k) was revealed using antibody staining (1:100 anti-H-2K^(k)(Becton Dickinson)+1:50 beriglobine (Aventis Behring) in PBS, 10 minutesat room temperature). The dead cells were stained with 0.25 μg/mlpropidiumjodide. The analysis was carried out in a flow-cytometer(FACScalibur, Becton Dickinson). Diagrams a) and b) show that usingelectrodes with doped synthetic material (PC/CF/Gr=polycarbonate+20%carbon fibers+15% graphite and PEEK-CF=polyetheretherketone+40% carbonfibers) at least similar results can be achieved in respect oftransfection efficiency compared to the use of electrodes made ofaluminium. Due to the significantly lower mortality rates if electrodesaccording to the invention are used, a significant advantage incomparison to the use of conventional electrodes is achieved because ofthe better ratio of transfection efficiency to mortality.

FIG. 6 shows diagrams of a flow-cytrometric analysis of transfected HL60cells (human lymphoma cells). Realisation and conditions of thisapproach correspond to that described in FIG. 5 with the exception thatduring electroporation the voltage pulses were modified (here: 1. Pulse:1000 V, 70 μs; 2. Pulse: U_(start)=81 V, 22 mC). In this approach,results could be achieved using electrodes made of doped syntheticmaterial, which are at least similar to those if electrodes made ofaluminium are used.

FIG. 7 shows diagrams of a flow-cytometric analysis of transfectedJurkat cells (human T-cell line) using electrodes according to theinvention made of polyamide 66 which was doped with 30% carbon fibers.Electroporation was carried out in 100 μl RPMI medium without phenolredwith 5 μg of the plasmid pEGFP-C1 by a pulse of 150 V and 5 μs followedby a pulse having a start voltage of 108 V and a charge of 80 mC. Theanalysis was carried out after 4 hours. Also in this example,transfection efficiency as well as survival rate could be significantlyincreased by the use of containers or electrodes made of doped syntheticmaterial according to the invention in comparison to conventionalelectrodes made of aluminium. Thus, the results depicted in FIGS. 5-7clearly prove that, compared to conventional cuvettes, inelectroporation the transfection efficiencies can be significantlyincreased and the mortality rate can be significantly decreased if thecontainers according to the invention are used. This can be primarilyexplained by the fact that, surprisingly, similar current flows can beensured using electrodes according to the invention while the knowndrawbacks due to the release of metal ions from the electrodes and hencetoxic effects on the cells can be avoided.

FIG. 8 shows diagrams of a flow-cytometric analysis of transfected HUVECcells (human umbilical cord vein endothelial cells) using electrodesmade of polyphenylensulfide with 40% carbon fibers according to theinvention compared to conventional electrodes made of aluminium.Transfection was performed in a cell specific medium including 5 μg/100μl plasmid DNA, wherein in this approach various voltage pulses wereapplied for compensation of the somewhat lower conductivity of the dopedsynthetic material (Pulse for PPS/CF: 1000 V, 100 ps; Pulse foraluminium: 500 V, 100 ps). After 60 hours incubation the cells wereflow-cytometrically tested for expression of a fluorescent protein. Deadcells were stained with 0.25 μg/ml propidiumjodide in this approach aswell. The results show that the containers according to the inventionare generally also suitable for primary human cells. Also in thisapproach, the ratio of transfection efficiency to mortality rate isbetter than with the use of conventional electrodes made of aluminium.This effect can be further enhanced by compensation of the effectivelyhigher resistance of the doped synthetic material by increasing theapplied voltage. By this measure, the electric conditions within thecell suspension can be adapted if electrodes made of doped syntheticmaterial are used and the transfection efficiency can be furtherincreased.

FIG. 9 shows micrographs of CHO cultures always two days afterelectroporation using electrodes made of aluminium (a) and electrodesmade of polyphenylensulfide including 40% carbon fibers (b). Also inthis approach, the somewhat lower conductivity of electrodes made ofdoped synthetic material was compensated by an increase of the first andsecond voltage pulses (PPS/CF: 1000 V, 100 ps and Ustar,=108 V, 100 mC;Aluminium: 500 V, 100 μs and U_(start)=76 V, 60 mC). Precipitates areclearly visible when electrodes made of aluminium are used, some of thembeing marked by inserted arrows in a). These particles precipitate ontothe cells. But those particles are not visible if electrodes made ofdoped synthetic material are used so that precipitation of particles canbe obviously avoided by the use of the container according to theinvention. This fact has a positive effect on the survival rate of thecells and it is also beneficial for the further handling of the cells.Medical compatibility of the electroporation products is hereby alsoenhanced so that, for example, the possible use of transfected primarycells for ex vivo gene therapy is affected particularly advantageous.

FIGS. 10 and 11 each show diagrams of a flow-cytometric analysis ofcells (FIG. 10: HUVEC cells, FIG. 11: CHO cells) which were incubated indifferently treated solutions. The containers according to the inventionwere filled with simple buffer solutions, such as PBS, without cells andexposed to a high voltage pulse three times in a row (Aluminium: 500 V,100 μs and U_(start)=115 V, 100 mC; doped synthetic material: 1000 V,100 μs and U_(start)=95 V, 112 mC) in order to analyse the effects ofpossible cell-damaging components which could be released during anelectric discharge from various electrodes made of doped syntheticmaterial and from electrodes made of aluminium and which in addition totheir direct effects reveal further effects on the cell culture justafter the pulses. Subsequently, 100 μl of the solutions pulsed by thevarious electrodes were added to 400 μl culture medium (EGM-2BulletKit/Clonetics). HUVEC cells (values after 18 hours: 5×10⁴ cells,values after 96 hours: 2.5×10⁴ cells) and CHO cells (values after 20hours: 10⁵ cells, values after 72 hours: 2×10⁴ cells), respectively,were transferred into 24-well plates in these media. The number of deadcells and living cells, respectively, was flow-cytometrically determinedafter various times of incubation at 37° C. and 5% CO₂. After detachingthe cells using 1 μg/ml trypsine in 1 mM Ethylendiaminthetraacetate inPBS and mixture of these cells with the culture supernatant, 0.25 μl/mlpropidiumjodide was added for determination of dead cells. For stainingof living cells, 0.2 μM carboxyfluoresceindiacetate-succinimdylester(CFDA-SE) in PBS +0.5% bovine serum albumin were added and incubated for2 minutes at room temperature before the flow-cytometric analysis. Inorder to determine the total number of cells in one probe a definednumber of Flow-Count Fluorosphere Beads (Beckman Coulter) was added,which can be distinguished from the cells in the FACS. The countednumber of cells could be extrapolated this way to the whole volume inone well. The results represented in FIGS. 10 an 11 show that there is aslight advantage with the use of electrodes made of doped syntheticmaterial if compared to electrodes made of aluminium. In particular,after 4 days the solutions pulsed using electrodes made of syntheticmaterial are more beneficial in respect of survival and growth of CHOcells. With HUVEC cells a positive effect of the plastic electrodes usedin comparison to conventional electrodes made of aluminium can bealready observed after 24 hours. Thus, these results are an indicationfor a better compatibility of electrodes made of doped syntheticmaterial in respect of the release of cell-damaging components, whereinthe negative effects after the current flow have been investigated inthis approach. Due to the avoidance of the release of toxic metal ions abetter biological compatibility of the containers according to theinvention is additionally achieved. Thus, these are primarily withrespect to the further use of transfected cells, for example the use oftransformed primary cells for ex vivo gene therapy, significantly moreadvantageous than conventional containers or cuvettes.

FIG. 12 shows a perspective representation of one possible embodiment ofa container according to the invention. The container 20 shown isgenerally formed like a conventional cuvette. The container is formed byan outer limit 21 which builds an inner chamber 22 which has thecapacity for receiving an aqueous solution. For example, cells,derivatives of cells, subcellular particles and/or vesicles can besuspended in this aqueous solution. In addition to the aqueous solutionor suspension, the container may also contain, for instance, adheringcells, derivatives of cells, subcellular particles and/or vesicles. Twoparallel electrodes 25, 26 are disposed in two parallel and oppositelyarranged sidewalls 23, 24 of the outer limit 21. Both electrodes 25, 26are made of a synthetic material which, according to the invention, isdoped with at least one conductive substance. The dope may consist of,for instance, carbon fibers, graphite, soot, carbon nanotubes or anintrinsically conductive synthetic material, or a combination of one orseveral of these substances as well. The outer limit 21 consists of antransparent plastic material which is not electroconductive. Due to theinjection-mouldability of all components the container 20 according tothe invention can be produced using two-component injection moulding. Inthis case, at first the outer limit 21 was injection-moulded using annon-conductive plastic. The doped synthetic material beinginjection-mouldable as well was injection-moulded into recessed windows(which are not visible any more) through injection moulding channels 27,28. Thus, a very simple and cost-effective production of the container20 according to the invention is possible. Such container 20 beingformed like a cuvette is primarily beneficial if it shall be used in aconventional electroporation device. However, depending on the kind ofapplication, the container according to the invention comprises allpossible embodiments that make sense anyway.

FIG. 13 shows a perspective representation (b) of a possible embodimentof a container according to the invention as well as a longitudinalsection (a) of the same. The container 30 shown is also formed like acuvette. The container is formed by an outer limit 31 which builds aninner chamber 32 which acts for receiving an aqueous solution. Twoparallel electrodes 33, 34 are disposed in two parallel and oppositelyarranged sidewalls 35, 36 of the outer limit 31. Both electrodes 33, 34consist of a synthetic material, for example polyamide 66 or polyamide6, which is doped with at least one conductive substance according tothe invention. In an advantageous embodiment of the invention the dopemay be, for instance, a mixture of carbon fibers and graphite. The outerlimit 31 consists of a transparent plastic material which isnon-electroconductive. Because of the injection-mouldability of allcomponents the container 30 according to the invention can be producedusing two-component injection moulding. Hence, at first the outer limit31 can be injection-moulded of a non-electroconductive plastic material.The doped synthetic material which is also injection-mouldable may besubsequently injection-moulded in recessed windows through injectionmoulding channels 37, 38. Thus, very simple and cost-effectiveproduction of the container 30 according to the invention is possiblethis way. An oblique part 39 is disposed in the lower half of thecontainer 30, which enables the container 30 to be adapted to thegeometry of the respective receiving element of the device used, i.e. anelectroporator. Additionally, the distance between the electrodes 33, 34may be varied by different designs of the oblique part 39 so that theforward resistance can be changed.

FIGS. 14 to 16 show diagrams of flow-cytometric analyses of transfectedcells using cuvettes having electrodes according to the inventionaccording to FIG. 13, which have a distance to each other of 1.5 mm.These containers are tested in direct comparison to cuvettes havingelectrodes made of aluminium with a distance between the electrodes of 2mm. Due to the 25% reduction of the distance between the electrodes thehigher resistance of the material of the doped plastic was compensatedin respect of aluminium in order to achieve effectively the sameconductivity per cross-sectional surface. The experimental procedurewith polymer cuvettes corresponds to that with aluminium, wherein anelectroporator having spring contacts made of brass (Nucleofector™ I,amaxa GmbH, Cologne) was used. Cell specific Nucleofector™ kits (amaxaGmbH, Cologne) were always used as solutions for receiving the cells.The cuvettes according to the invention always comprise electrodesconsisting of polyamide (PA6 or PA66) doped with about 38-42% w/w carbonfibers and about 33-37% w/w graphite (overall concentration of the dope:about 70-80 % wlw).

FIG. 14 shows diagrams of a flow-cytometric analysis of HL-60 cells(human lymphoma cells) after electroporation using polymer electrodes(PA6) according to the invention compared to electrodes made ofaluminium. 2 μg pEGFP-C1 DNA (Clontech) were added to each probe of 100μl solution including 10⁶ HL-60 cells (ATCC). Two voltage pulses (1000V, 100 μs and U_(start)=90 V, 75 mC) were applied to various cuvettescontaining these probes which were then immediately incubated inIscove's modified Dulbecco's medium containing L-glutamine and 20% fetalcalf serum (Gibco) in an incubator at 37° C./5% CO₂. After 24 hours thecells were harvested and propidiumjodide as well as 25.000 APC-markedbeads (Becton Dickinson) were added to each probe. Determination ofpropidiumjodide-stained cells and transfected cells based on theabsolute number of cells per probe in a flow-cytometer (FASCalibur,Becton Dickinson) was possible this way. It turned out herein that thetransfection efficiency could be significantly increased and that themortality rate could be slightly decreased by the use of cuvettesaccording to the invention in comparison to conventional cuveftes havingelectrodes made of aluminium.

FIG. 15 shows diagrams of a flow-cytometric analysis of CD3⁺ T-cellsafter electroporation using polymer electrodes (PA6) according to theinvention in comparison to electrodes made of aluminium. 2 μg pH-2K^(K)(mouse MHC I heavy chain) were added to each probe of 100 μl solutioncontaining 5×10⁶ freshly isolated PBMC. Two voltage pulses (1000 V, 100μs and U_(start)=96 V, 56 mC) were applied without interruption tovarious cuvettes containing these probes which are then directlyincubated in AIM-V medium containing 10% fetal calf serum (Gibco) in anincubator at 37+ C./5% CO₂. After 24 hours the cells were harvested andpropidiumjodide as well as 25.000 APC-marked beads (Becton Dickinson)per probe were added. Additionally, the cells were stained using thefluorescein-isothiocyanate-stained anti-H-2K^(K) antibody (BectonDickinson) and an antibody against the human T-cell-specific CD3 antigenwhich is coupled to APC (Becton Dickinson). Determination ofpropidiumjodide-stained cells and transfected T-cells based on theabsolute number of cells per probe in a flow-cytometer (FASCalibur,Becton Dickinson) was hereby possible. Approximately similar resultscould be achieved this way.

FIG. 16 shows diagrams of a flow-cytometric analysis of human umbilicalcord vein endothelial cells (HUVEC) after electroporation using polymerelectrodes (PA6) according to the invention in comparison to electrodesmade of aluminium. 2 μg pEGFP-C1 DNA (Clontech) was added to each probeof 100 μl solution containing 6.8'10⁵ HUVEC cells. A voltage pulse (1000V, 100 μs) was applied to various cuvettes containing these probes whichwere then directly incubated in EGM-2 medium for endothelial cells(Clonetics) in an incubator at 37° C./5% CO₂. After 24 hours the cellswere harvested and porpidiumjodide as well as 25.000 APC-marked beads(Becton Dickinson) were added to each probe. Determination ofpropidiumjodide-stained cells and transfected cells based on theabsolute number of cells per probe in a flow-cytometer (FASCalibur,Becton Dickinson) was possible this way. It turned out herein that withthe use of cuvettes according to the invention instead of conventionalcuvettes having electrodes made of aluminium, the transfectionefficiency could be increased and the mortality rate could be decreased.

FIG. 17 shows diagrams of a flow-cytometric analysis of HL-60 cells(human lymphoma cells) 24 and 96 hours after electroporation usingpolymer electrodes (PA66 and PA6) according to the invention incomparison to electrodes made of aluminium. Conditions and proceduresgenerally correspond to those described in FIG. 14 with the exceptionthat the cuveftes made of aluminium as well as the cuvettes according tothe invention have a distance between the electrodes of 2 mm. Thecuvettes used herein each further have electrodes made of polyamidedoped with about 33-37% w/w carbon fibers and about 23-27% w/w graphite(overall concentration of the dope: about 55-65% w/w). In order tocompensate the higher resistance of the polymer electrodes differentpulse parameters are used herein (Synthetic material: 1000 V, 100 μs andU_(start)=102 V, 75 mC and Aluminium: 800 V, 100 μs and U_(start)=90 V,75 mC). Also in this approach, the transfection efficiency could beslightly increased and the mortality rate could be slightly decreased bythe use of polymer electrodes according to the invention.

List of Abbreviations Used:

-   A Ampere-   C Coulomb-   CHO Chinese hamster ovary-   cm Centimeter-   DNA Deoxyribonucleic acid-   Gew.-% Percent by weight-   h Hours-   HL-60 Human lymphoma 60-   HUVEC Human umbilical cord vein endothelial cells-   kV Kilovolt-   mC Millicoulomb-   mM Millimolar-   ms Milliseconds-   PA Polyamide-   PBMC Peripheral blood mononuclear cells-   PBS Phosphate-buffered saline-   pH Negative logarithm of the hydrogen-ion concentration-   PJ Propidiumjodide-   RNA Ribonucleic acid-   RPMI Rosewell Park Memorial Institute-   μg Microgram-   μl Microliter-   μs Microseconds-   U Voltage-   U_(Anfang) Voltage_(start)-   V Volt

List of Reference Numbers:

-   1 Experimental assembly-   2 Spacer plate-   3 Electrode-   4 Electrode-   5 Recess-   6 Copper plate-   7 Copper plate-   8 Threaded part-   9 Threaded part-   10 Innerspace-   11 Inneredges-   12 Experimental assembly-   13 Copper wire-   14 Copper wire-   20 Container-   21 Outer limit-   22 Inner chamber-   23 Sidewall-   24 Sidewall-   25 Electrode-   26 Electrode-   27 Injection moulding channel-   28 Injection moulding channel-   30 Container-   31 Outer limit-   32 Inner chamber-   33 Electrode-   34 Electrode-   35 Sidewall-   36 Sidewall-   37 Injection moulding channel-   38 Injection moulding channel-   39 Oblique part

1. Container for receiving an aqueous solution-, which is formed atleast partially by an outer limit which forms an inner chamber forreceiving said solution, and which comprises at least one area whichacts as an electrode when an electric voltage is applied and asubsequent discharge occurs, wherein said at least one electrode is madeof a conductive synthetic material which is, or is at least based on, aplastic material which is doped with at least one conductive substance,and wherein the overall concentration of said dope in said plasticmaterial is 40-80% w/w.
 2. Container according to claim 1, wherein saiddope consists essentially of carbon fibers, graphite, soot and/or carbonnanotubes.
 3. Container according to claim 1, wherein the overallconcentration of said dope in said plastic material is 40-60% w/w,preferably 50-60% w/w, in particular 55-60% w/w.
 4. Container accordingto claim 1, wherein the overall concentration of said dope in saidplastic material is 50-80% w/w, preferably 60-80% w/w, most preferred70-80% w/w, in particular 74-76% w/w.
 5. Container according to claim 1,wherein said plastic material is polycarbonate, polyetheretherketone,polypropylene, polyamide, polyphenylensulfide or a mixture of thesepolymers, or at least based on one or several of these polymers, and/orwherein said plastic material is an intrinsically conductive syntheticmaterial.
 6. Container according claim 5, wherein said intrinsicallyconductive synthetic material is polyaniline, polyacetylene,poly-para-phenylene, poly-para-phenylensulfide, polypyrroles,polythiophene, polypropylene, or at least based on one or several ofthese polymers.
 7. Container according to claim 1, wherein said outerlimit is made of synthetic material.
 8. Container according to claim 7,wherein said synthetic material is the same plastic material as theplastic material on which said at least one electrode is based. 9.Container according to claim 1, wherein said at least one electrode isintegrated into said outer limit.
 10. Container according to claim 1comprising at least two electrodes being made of the same material. 11.Container according to claim 1 comprising at least two electrodes,wherein said at least two electrodes are made of different materials.12. Container according to claim 1, wherein said at least one electrodeis made of polyamide, in particular polyamide 66 or polyamide 6, dopedwith 25-45% w/w, preferably 30-40% w/w, in particular 33-37% w/w, carbonfibers and 15-35% w/w, preferably 20-30% w/w, in particular 23-27% w/w,graphite.
 13. Container according to claim 1, wherein said at least oneelectrode is made of polyamide, in particular polyamide 66 or polyamide6, doped with 30-50% w/w, preferably 35-45% w/w, in particular 39-41%w/w, carbon fibers and 25-45% w/w, preferably 30-40% w/w, in particular34-36% w/w, graphite.
 14. Container according to claim 1, wherein saidat least one electrode is made of polycarbonate doped with 15-40% w/wcarbon fibers and 1-40% w/w graphite.
 15. Container according to claim1, wherein said at least one electrode is made of polyetheretherketonedoped with 40-50% w/w carbon fibers.
 16. Container according to claim 1,wherein said at least one electrode is made of polyamide, preferablypolyamide 66, doped with 40% w/w carbon fibers.
 17. Container accordingto claim 1, wherein said at least one electrode is made of polypropylenedoped with 40% w/w carbon fibers.
 18. Container according to claim 1,wherein said at least one electrode is made of polyphenylensulfide dopedwith 40-50% w/w carbon fibers.
 19. Container according to claim 1,wherein said outer limit comprises at least one opening for supplyingsaid solution and at least one opening for draining off said solution.20. Container arrangement comprising at least two, preferably 6, 12, 24,48, 96 or more, containers according to claim 1 being joined to buildone unit.
 21. Method for producing containers or container arrangementsaccording to claim 1, by two-component injection moulding comprising:(a) at first injection-moulding the outer limit so as to leave onerecessed window, and (b) subsequently injection-moulding the conductivesynthetic material made of doped plastic into said at least one window,or alternatively (a) at first injection-moulding said at least oneelectrode is from said doped plastic material, and (b) subsequentlyinjection-moulding said outer limit around said at least one electrode22. Method for treatment of cells, derivatives of cells, subcellularparticles and/or vesicles by means of electric current comprising: a)transferring said cells, derivatives of cells, subcellular particlesand/or vesicles into an inner chamber of at least one containeraccording to claim 1 comprising and at least one further electrode, andb.) applying voltage to said electrodes and generating a current flow insaid inner chamber of said container.
 23. Method according to claim 22,wherein said electric current reaches a current density up to 120 A/cm²,preferably 80 A/cm².
 24. Method according to claim 22, whereinbiologically active molecules are solved in said solution, and transferof said biologically active molecules into living cells is achieved ofvia a voltage pulse having a field strength of 2 to 10 kV*cm⁻¹ and aduration of 10 to 200 μs.
 25. Method according to claim 24, wherein saidtransfer of said biologically active molecules into said cells isachieved by a current flow following said voltage pulse withoutinterruption, having a current density of 2 to 14 A*cm⁻², preferably 5A*cm⁻², and a duration of 1 to 100 ms, preferably 50 ms.
 26. Containeraccording to claim 1, wherein said aqueous solution comprises cells,derivatives of cells, subcellular particles and/or vesicles. 27.Container according to claim 7, wherein said synthetic material is atransparent plastic material.
 28. Method according to claim 22, whereinsaid cells, subcellular particles and or vesicles are transferred intoinner chambers of at least two containers.
 29. Method according to claim24, wherein said biologically active molecules are nucleic acids.